Col6a1

Manufactured by Santa Cruz Biotechnology

Sourced in United Kingdom

COL6A1 is a gene that encodes the alpha-1 chain of type VI collagen. Type VI collagen is a component of the extracellular matrix and plays a role in cell-matrix interactions. The COL6A1 protein is involved in the assembly and stabilization of type VI collagen fibrils.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions) Mmp 1, by Santa Cruz Biotechnology (1 mentions) Mini protean tgx stain free protein gel, by Bio-Rad (1 mentions) Timp 2, by Abcam (1 mentions) Goat anti mouse hrp, by Merck Group (1 mentions) Thbs1, by Santa Cruz Biotechnology (1 mentions) Criterion stain free imaging system, by Bio-Rad (1 mentions) Serpine1, by Santa Cruz Biotechnology (1 mentions) Ultrascence pico ultra western substrate, by GeneDireX (1 mentions) Goat anti rabbit hrp, by Merck Group (1 mentions) Chemidoc mp imaging system, by Bio-Rad (1 mentions) Trans blot turbo system, by Bio-Rad (1 mentions) Dapi solution, by Thermo Fisher Scientific (1 mentions) P4ha1, by Abcam (1 mentions) Alexa fluor 488, by Abcam (1 mentions) Alexa fluor 594, by Abcam (1 mentions) Alexa fluor 488, by Cell Signaling Technology (1 mentions) Aggrecan, by Santa Cruz Biotechnology (1 mentions) Col2a1, by Abcam (1 mentions) Aggrecan, by Thermo Fisher Scientific (1 mentions)

6 protocols using col6a1

Protein Expression Analysis Workflow

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CM were mixed with 4× Laemmli sample buffer (containing 2-mercaptoethanol) and boiled for 10 min at 95°C. 15-μl protein volumes were separated on 4–15% Mini-PROTEAN TGX stain-free protein gels (4568086; Bio-Rad), and whole proteins were detected with the Criterion Stain-free imaging system (Bio-Rad). Next, the proteins were transferred on polyvinylidene difluoride membranes using the Trans-Blot-Turbo system (Bio-Rad). The membranes were blocked in 5% nonfat dry milk/TBS-T for 1 h at RT. The following primary antibodies were diluted 1:1,000 in 5% BSA/TBS-T and incubated overnight at 4°C: MMP1 (sc-30069; Santa Cruz), SERPINE1 (sc-5297; Santa Cruz), TIMP2 (ab53730; Abcam), COL6A1 (sc-377143; Santa Cruz), FN1 (F3648; Sigma-Aldrich), and THBS1 (sc-59887; Santa Cruz). Membranes were 3× washed in TBS-T and the following secondary antibodies were diluted 1:20,000 in 2.5% nonfat dry milk/TBS-T and incubated for 1 h at RT: goat-anti-mouse-HRP (SAB3701073-2; Sigma-Aldrich) or goat-anti-rabbit-HRP (SAB3700878-1; Sigma-Aldrich). HRP was visualized by the UltraScence Pico Ultra Western Substrate (CCH345-B; GeneDireX) and the ChemiDoc MP imaging system (Bio-Rad).

Blache U., Horton E.R., Xia T., Schoof E.M., Blicher L.H., Schönenberger A., Snedeker J.G., Martin I., Erler J.T, & Ehrbar M. (2019). Mesenchymal stromal cell activation by breast cancer secretomes in bioengineered 3D microenvironments. Life Science Alliance, 2(3), e201900304.

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Tissue Immunofluorescence Staining Protocol

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ECs derived from GSCs were incubated with antibodies against P4HA1, CD31, and COL6A1, followed by Alexa Fluor 488 and Alexa Fluor 594 conjugated secondary antibodies (Cell Signaling Technology, USA) to visualize the primary antibodies complexes. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI, ThermoFisher Scientific, USA).
Human and mouse glioma tissue samples were fixed in 4% formalin solution, embedded in paraffin, and cut into 4 um sections for tissue slice immunofluorescence. After dewaxing, the tissue sections were immersed in citric acid solution and heated in a microwave oven (Siemens, Germany) for antigen retrieval. The tissue sections were incubated overnight at 4°C with antibodies to P4HA1 (1:200, Abcam, Cambridge, UK), COL6A1 (1:50, Santa Cruz, USA) and CD31 (1:400, Abcam, Cambridge, UK), followed by Alexa Fluor 488, Alexa Fluor 594 and Alexa Fluor 594 conjugated secondary antibodies. DAPI solution (ThermoFisher Scientific, USA) labelled cell nuclei. Images were captured in black and white mode by the corresponding channel in the Zeiss orthographic microscope and then colored.

Han X., Wang Q., Fang S., Wang J., Liu F., Zhang J, & Jin G. (2022). P4HA1 Regulates CD31 via COL6A1 in the Transition of Glioblastoma Stem-Like Cells to Tumor Endothelioid Cells. Frontiers in Oncology, 12, 836511.

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Immunohistochemical Analysis of hMSC Pellets

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hMSC pellets were fixed in 4% paraformaldehyde (Sigma, St Louis, MO) and embedded in paraffin. Pellet sections (10 μm) were permeabilized in cold methanol (Sigma, St Louis, MO), before blocking in PBS containing 1% BSA, 10% FBS, and 0.4% Triton X‐100 and then incubated with the following primary antibodies overnight: rabbit polyclonal Col2a1 (Abcam, Cambridge, MA 34712; 1:100), Aggrecan (1:500), rabbit polyclonal Col6a1 (Santa Cruz, Santa Cruz, CA 20649, 1:100) at the specified dilutions. The antibody anti‐Aggrecan used for this study was a kind gift from Prof. R. L. Smith.16 The following day, cells were washed in PBS and incubated for 1 hr in secondary antibody (Alexa 594 goat anti‐rabbit 1:250, Invitrogen, Carlsband, CA) and cellular DNA was counterstained with DAPI (Life Technologies, Carlsband, CA).

Smeriglio P., Lee J, & Bhutani N. (2017). Soluble Collagen VI treatment enhances mesenchymal stem cells expansion for engineering cartilage. Bioengineering & Translational Medicine, 2(3), 278-284.

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Western Blot Analysis of EMT Markers

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Cells were washed twice with cold PBS and were lysed in RIPA buffer with Phenylmethanesulfonyl fluoride (both Beyotime). BCA Protein Assay Kit (Beyotime) was used to quantify protein concentrations. Proteins were separated by SDS-PAGE and transferred onto PVDF membranes (Immobilon-P). 5% bovine serum albumin was performed to block membranes. Primary antibodies include Fzd7 (Santa Cruz Biotechnology, sc-293,261), Col6a1 (Santa Cruz Biotechnology, sc-377,143), Wnt5a/b (Cell Signaling Technology, #2530), IL-6 (Cell Signaling Technology, #12153), p-Stat3 (Cell Signaling Technology, #9145), E-cadherin (Cell Signaling Technology, #3195), Vimentin (Cell Signaling Technology, #5741), Slug (Cell Signaling Technology, #9585), Zeb1 (Sigma, SAB2102759), Smad3 (Cell Signaling Technology, #9523), Yap1 (Cell Signaling Technology, #14074), CD44 (Abcam, ab51037) and GAPDH (Proteintech, HRP-60004). Membranes were incubated with primary antibody overnight, and with horseradish peroxidase–conjugated secondary antibody for 2 h. SuperSignal Chemiluminescent Substrates (Thermo Fisher Scientific) and imaging systems were used to analysis the results.

Yin P., Bai Y., Wang Z., Sun Y., Gao J., Na L., Zhang Z., Wang W, & Zhao C. (2020). Non-canonical Fzd7 signaling contributes to breast cancer mesenchymal-like stemness involving Col6a1. Cell Communication and Signaling : CCS, 18, 143.

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Immunohistochemical Analysis of Human Breast Cancer

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Immunohistochemistry (IHC) analysis was performed on 4 μm paraffin sections of human BC tissues and mouse xenograft tumors. Xylene and gradient alcohols were performed to deparaffinize and hydrate, respectively. 3% H₂O₂ was added to the sections to remove endogenous peroxidase. Sections were incubated with citrate buffer to repair antigen, and blocked by BSA. Primary antibodies were used as follows: Fzd7 (Santa Cruz Biotechnology, sc-293,261), Col6a1 (Santa Cruz Biotechnology, sc-377,143), Ki67 (Invitrogen, 14,569,982), Cleaved caspase-3 (Cell Signaling Technology, #9661). After incubated with primary antibodies overnight at 4 °C in a wet box, biotinylated secondary antibodies were added. Diaminobenzidine (BOSTER) was used to stain the sections dissolved in Tris-HCl and H₂O₂. Then sections were re-stained in hematoxylin, dehydrated with gradient alcohol and xylene and sealed with cover slides.

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Protein expression analysis by Western blot

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Tissue samples and cell cultures were lysed as described elsewhere. 5 (link) Protein samples (20-50 μg) were run on SDS-PAGE and Western blot was performed. The membranes were probed with antibodies against: 1:500 COL1A1 (No. SAB1402151, Sigma-Aldrich), 1:500 COL3A1 (sc-28888, Santa Cruz Biotechnology), 1:500 COL6A1 (sc-377143, Santa Cruz Biotechnology) and β-actin or β-tubulin as loading controls (Sigma-Aldrich).

Bertorello A.M., Pires N., Igreja B., Pinho M.J., Vorkapic E., Wågsäter D., Wikström J., Behrendt M., Hamsten A., Eriksson P., Soares-da-Silva P, & Brion L. (2015). Increased arterial blood pressure and vascular remodeling in mice lacking salt-inducible kinase 1 (SIK1). Circulation research, 116(4).

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